This is a competitive renewal of NIH grant DK082659 entitled Developmental Genes, miRNAs and Adipose Tissue. Over the past decade it has become clear that adipose tissue is more complex than originally believed. In addition to both white and brown fat, both types of fat are heterogeneous. Accumulation of visceral WAT is associated with insulin resistance and metabolic disease, whereas accumulation of subcutaneous WAT does not have such a risk and may even be protective. This is due, at least in part, to cell-autonomous, functional differences between adipocytes in these depots. During the past grant period, we have shown that white adipocytes and preadipocytes from different fat depots exhibit markedly different levels of expression of developmental/patterning genes, suggesting developmental heterogeneity of WAT. We have focused on three of these genes (Shox2, Tbx15 and Glypican-4), since in humans, the expression of these genes strongly correlates with BMI and fat distribution. We have shown that overexpression or knockdown of these genes can alter adipocyte differentiation and function. For example, Shox2 regulates the lipolytic response of adipocytes, whereas Tbx15 can modulate mitochondrial metabolism and the balance between glycolytic and oxidative metabolism in fat cells. Glypican-4 (Gpc4), on the other hand, modulates insulin receptor binding and action, and is released from fat cells into the circulation where is can act on other target cells. We have found that different WAT depots also differ in their expression of miRNAs and that there are alterations in miRNAs in obesity and aging. The latter is due to a decrease in the expression of the miRNA processing enzyme Dicer. In exciting new data, we also show that adipose tissue is a major source of circulating miRNAs. These data have led to two inter-related hypotheses. First, we hypothesize that, in analogy to skeletal muscle cells or lymphocytes which look alike but have different functions, white adipocytes are heterogeneous in nature and that development/patterning genes contribute to this heterogeneity and program differences in adipose tissue development and function. Secondly, adipocyte function and heterogeneity is further modified by differences in miRNA expression. In addition these miRNAs are released into the circulation where they act as novel mediators of the effects of fat on other tissues. The specific aims going forward are to: 1) Determine how the developmental geneTbx15 contributes to heterogeneity of WAT and affects adipocyte determination and function. 2) Further characterize the role of Gpc4 as a modifier of adipocyte function and insulin signaling, and how Gpc4 is released from adipocytes into the circulation and contributes to changes in insulin sensitivity. 3) Determine how miRNA expression differs between adipocytes and preadipocytes from different depots, and how changes in miRNA expression affect function of adipocytes in different depots. We will also explore our preliminary findings that adipose tissue is a major source of circulating miRNAs and that these circulating miRNAs may serve as mediators of adipose effects on other tissues.